DESCRIPTION: A central question in biology is how a cell efficiently integrates the large number of pathways that comprise intermediary metabolism. Although it is clear that some metabolites are generated in a regulated manner to maintain physiological balance, it is less clear how the cell achieves efficient synthesis of molecules whose steady intracellular level is very low, such as coenzymes and their precursors. The concept of channeling metabolites through multienzymatic complexes (a.k.a. metabolons) is particularly attractive to the analysis of major biosynthetic pathways such as the one synthesizing adenosyl-cobalamin (Ado-CBL), because this important coenzyme is made in very small amounts. We are using the late steps of Ado-CBL biosynthesis as a model system for probing the metabolon hypothesis. We have previously reported genetic evidence suggesting that enzymes catalyzing these steps may interact, and that these interactions may indeed be needed for metabolite channeling. Towards that goal we have established the biochemical role of 3 of the 4 enzymes thought to comprise the metabolon, and initiated collaborative work with Dr. Ivan Rayment (Enzyme Institute, UW-Madison) to pursue the crystallographic analysis of these proteins. Work represented in this proposal will continue the biochemical characterization of these enzymes. Our multidisciplinary approach to understanding the cobalamin biosynthetic pathway will make important contributions to our knowledge of broad, basic biochemical concepts such as protein/protein interactions, protein/coenzyme interactions, enzymology, multienzyme complex formation and metabolite channeling. This basic knowledge will help us explain in molecular terms the complex phenotypes of mutants deficient in cobalamin synthesis and the impact of the cobalamin biosynthetic pathway on cell physiology.